Estudiantes eliminated by Cerro Porteno
Fellow Argentine side Velez Sarsfield qualified comfortably for the quarter-finals 5-0 on aggregate after a 2-0 away win over LDU Quito in the second leg of their first knockout round tie.
Despite their poor form in the Mexican championship, Chiapas came from behind three times to draw 3-3 with Colombia's Atletico Junior in Barranquilla and advanced on the away goals rule 4-4 on aggregate.
With captain Juan Sebastian Veron missing through illness, Estudiantes again drew 0-0 with Cerro before the Paraguayan side triumphed 5-3 on penalties. There is no extra-time in the competition.
Goalkeeper Diego Barreto saved the Argentine side's fourth penalty from Facundo Roncaglia before Javier Villarreal converted Cerro's fifth.
Estudiantes have suffered an alarming loss of form having gone eight matches in the Argentine Clausura championship and the Libertadores Cup without a win.
At high altitude in Quito, after an interruption of 17 minutes due to a lighting failure, LDU hit the woodwork twice in a brief spell of complete dominance.
LDU striker Hernan Barcos shot against the bar and less than two minutes later Velez keeper Marcelo Barovero saved from midfielder Luis Bolanos and Enrique Vera hit the rebound against the post.
Velez stunned the Ecuadorean side before halftime with a goal from midfielder Ricky Alvarez, who took a pass from substitute David Ramirez, rounded a defender and beat goalkeeper Alexander Dominguez.
Ramirez delivered another fine pass 10 minutes from time for substitute Ivan Bella to score Velez's second, tucking the ball behind Dominguez from a tight angle.
LDU defender Jorge Guagua was sent off in the dying minutes and Velez midfielder Augusto Fernandez came off after the final whistle with a wound near his left eye from an object thrown from the crowd.
Colombia striker Jackson Martinez did not make a popular return to his home country, scoring two equalisers for Chiapas at the Metropolitano on the Caribbean coast.
Chiapas, held 1-1 at home in last week's first leg, scored their crucial third equaliser with a shot from outside the box four minutes from time through midfielder Edgar Andrade.
The teams were both down to 10 men at that stage following red cards for Martinez and Junior left-back Cesar Fawcett for fighting in the 78th minute.